A recent outbreak of carbapenem resistant Enterobacteriaceae (CRE) associated with contaminated duodenoscopes at the Ronald Reagan UCLA Medical Center highlights the importance of early detection of these clinically significant pathogens. Coincidentally, the Obama Administration released The National Action Plan to Combat Antibiotic-Resistant Bacteria for which one of the steps of the action plan is to slow the emergence of resistant bacteria and prevent the spread of resistant infections. Detection and identification of carbapenemase producing organisms (CPO) in health-care centers is the first step to confining the source and preventing the potential spread of these multidrug resistant pathogens. However, detection of CPO in clinical laboratories is challenging, as isolates may only have moderate reductions in susceptibilities to carbapenems and resistance may be mediated by other mechanisms (i.e., chromosomal and plasmid-mediated cephalosporinases or extended-spectrum ?-lactamase (ESBL) producers with decreased membrane permeability). Molecular methods for the detection of carbapenemase genes are limited by the number of carbapenemase targets assayed in addition to other limitations. The reference phenotypic method for detection of carbapenemase-producing Enterobacteriaceae, the Modified Hodge test, suffers from a long turn- around time, lacks specificity and has poor sensitivity for certain types of carbapenemase enzymes (i.e., metallo-?-lactamase enzymes). This project aims to develop a low cost, easy-to-use (essentially one manual step), and rapid (10-15 min) assay to identify carbapenemase production in carbapenem resistant Gram-negative bacteria suitable for surveillance and clinical laboratory use. The proposed assay uses a luciferin derivatized ?-lactamase substrate for detection of ?-lactamase activity. All reagents will be formulated in a master mix (Reagent I) such that the assay involves essentially one manual step: addition of the sample to the master mix. To differentiate carbapenemases from other ?-lactamases, a second master mix containing a carbapenem will be formulated (Reagent II). The signal of Reagent I indicates the presence or absence of ?-lactamase while the signal ratio of Reagent II to Reagent I indicates whether the ?-lactamase is a carbapenemase. The proposed Phase I project will optimize the assay and use well-characterized isolates as well as prospective clinical isolates to evaluate the assay. Successful completion of this Phase I project will pave the way for a Phase II study, which will involve multiple sites and substantially larger sample numbers. A simple and rapid assay for detection of CPO will play a significant role in surveillance and help guide therapy and infection control measures.